The RT Biochemistry Section seeks to understand, through protein and nucleic acid mutagenesis, how HIV-1 reverse transcriptase (RT) interacts with the conformationally distinct nucleic acid duplexes encountered while converting the single-stranded RNA genome into an integration-competent double-stranded DNA. Using a combination of methods, we are investigating the synthetic (RNA- and DNA-dependent polymerase) and degradative [ribonuclease H (RNase H)] properties of this multifunctional retroviral enzyme, which continues to be a major target for development of therapeutic agents to inhibit HIV-1 replication and stem the progression of AIDS. (+) Strand DNA synthesis in retroviruses and LTR-containing retrotransposons initiates from the polyrpurine tract (PPT), the precision of which is critical to the integrity of the 5' LTR and its recognition by the viral integration machinery. Although PPT utilization has been studied with respect to alterations in RT or the sequence of the element, the molecular basis underlying this event remains obscure. Two recent studies from the RT Biochemistry Section have shed light on this processing, suggesting that the PPT actively participates in its processing by sequestering RT in an orientation placing the RNase H catalytic center over the biologically relevant processing site. The use of modified nucleosides allows the manner in which RT processes specialized substrates encountered during replication to be examined in detail. We evaluated structural features of the HIV-1 PPT by KMnO4 footprinting and site-directed mutagenesis, and processing of the Saccharomyces cerevisiae retrotransposon Ty3 PPT by targeted introduction of the non-hydrogen-bonding thymine isostere 2,4-difluorotoluene (F). Both studies suggest that structural features of the PPT sequester the polymerizing machinery in an orientation placing its RNase H catalytic center over the biologically relevant cleavage site. Future projects will extend the nucleoside analog strategy to the HIV-1 PPT to include placement of purine analogs in the RNA primer, and non-hydrogen-bonding T and C analogs (F and D, respectively) into the DNA template. Conformationally restricted RNA/DNA hybrids, containing locked nucleic acid (LNA)-inducing nucleosides in the RNA primer, will be investigated. Finally, the structure of the HIV-1 and Ty3 PPTs will be investigated using 19F-NMR and fluorine-substituted nucleoside analogs.